Investigation of the Ukrainian Cercospora beticola isolates

INVESTIGATION OF THE UKRAINIAN CERCOSPORA BETICOLA ISOLATES

Výzkum izolátů Cercospora beticola na Ukrajině

A. Syumka

Institute for Sugar Beet Research of UAAS, Ukraine

Summary: Significant differences among isolates, as measured by disease severity on sugar beet cultivars. Studied defined medium conditions for colony growth and for maximum and minimum cercosporin accumulation in isolates of C. beticola. However, the present study did identify certain Ukrainian isolates for future investigation of cercosporine production. Our data also show that screening isolates of C. beticola for cercosporin production under cultural conditions is unreliable and question the reliability of correlating toxin production in vitro to the virulence of a Cercospora isolate.

Key words: Cercospora leaf spot disease, Cercospora beticola isolates, pathogenicity specialization, mycelial growth, cercosporin production

Souhrn: Znalost fyziologické a biochemické specializace patogena je základním požadavkem programu šlechtění rezistentních odrůd cukrovky. Byly zjištěny průkazné rozdíly mezi izoláty Cercospora beticola, měřené jako síla napadení odrůd (hodnoceno 7 izolátů infikované cukrovky z různých regionů řepařské zóny Ukrajiny). Byly zkoumány podmínky definovaného media pro růst kolonie a pro maximální a minimální akumulaci cercosporinu v izolátech C. beticola. Nicméně předložená studie identifikovala jisté ukrajinské izoláty pro budoucí výzkum produkce cercosporinu. Naše údaje rovněž ukazují, že prověřování izolátů pro produkci cercosporinu v pěstebních podmínkách je nespolehlivé a otázkou je spolehlivost produkce korelujícího toxinu in vitro k virulenci izolátu Cercospory.

Klíčová slova: Cercosporová listová skvrnitost, izoláty Cercospora beticola, specializace patogenicity, růst mycelia, produkce cercosporinu

Introduction

Knowledge of physiologic and biochemical specialization of a pathogen is a basic requirement for a variable program of breeding disease-resistant cultivars. Cultural variation and virulence generally were the criteria used to differentiate races [4, 5]. Studies on the cercosporin toxin produced by Cercospora isolates have documented and important role for this toxin in pathogenesis of host plants [2].

Material and methods

Isolates of Cercospora beticola: All 7 isolates were isolated in 2002 from infected of sugar beets grown in different regions of the Ukrainian sugar beet zone.

Culture media [1]: Beet leaf dextrose medium contained 100 ml beet leaf extract, 20 g dextrose, and 15 g agar per liter. Malt medium contained 15 g of malt extract, 3 g of peptone, 30 g of glucose, and 15 g of agar per liter. Synthetic agar medium - composition of this medium was as follows: MgSO4 7H2O, 0.5 g; FeCl3, 0.2 mg; ZnSO4 7H2O, 0.2 mg; MnSO4 4H2O, 0.1 mg; KH2PO4, 0.908 g; K2HPO4, 1.160 g; NaNO3, 1.0 g; dextrose, 20 g; Difco yeast extract, 1.0 g; and agar, 15 g per litre.

Growth conditions: The effects of various treatments on growth, colony weight and cercosporin production were studied by culturing the fungus on 10 ml nutrient agar in Petri plates. The fungi cultures were incubated at 25 ± 10°C. The light source consisted of cool-white fluorescent lamps.

Cercosporin assay: Cercosporin was extracted by soaking in 5 N KOH in the dark for 4 hr, after which absorbance of the soaking solution was measured at 480 nm in a spectrophotometer [3].

Pathogenicity analysis: The sugar beet cultivars - highly susceptible and highly resistant to C. beticola - hybrid Ukrainian MS 70, Ivanivskyi MS 33, Yaltushkivskyi MS 72 and variety Yaltushkivskyi 64 were pathogenicity tested by inoculated from Cercospora isolates. Conidia and mycelium were atomized onto the foliage until all leaves were thoroughly wetted. Disease ratings of 0 to 5 were made every 14 days after inoculation. A randomized block design with four replications was used.

Results

Cercospora isolates colony area and dry colony weight were not associated for Malt medium (r= +0,13) but was associated for Beet leaf dextrose (r= +0,7) and Synthetic medium (r= +0,63).

Table 1: Mean dry weight (g) of mycelium for 20 days for 7 isolates of Cercospora beticola

Growth media had large effects on the production of cercosporin in agar cultures of the Cercospora isolates tested, and these effects differed among isolates. The best media for cercosporin production in most of the isolates were Malt and Synthetic agar medium.

Table 2: Mean disease reaction on sugar beet lines inoculated with 4 isolates of Cercospora beticola

a Control inoculated with sterile water

b Disease ratings based on a scale of 0 to 5, with 0 = no apparent infection and 5 = complete defoliation

Table 3: Production of cercosporin (nmol per plug) by 7 isolates on 3 media

Correlation studies indicated that cercosporin production in culture and disease severities in field pathogenicity test were associated; however, cercosporin production in isolate - medium test was associated with media effects.

Conclusion

Results demonstrated the variability among isolates of Cercospora beticola. Significant differences among isolates, as measured by disease severity on sugar beet cultivars (table 2). Table 3 shows that various isolates of C. beticola differ greatly in ability to produce cercosporin. It is interesting to note that the most active cercosporin producers were the isolates C-3 and C-6, which show the biggest pathogenicity effects from plants growing in the field.

References

1. BALIS C., PAYNE M.G. Triglycerides and cercosporin from Cercospora beticola: fungal growth and cercosporin production //Phytopathology. – 1971. – Vol.61. – P. 1477-1484.

2. DAUB M.E., EHRENSHAFT M. The photoactivated Cercospora toxin cercosporin: contributions to plant disease and fundamental biology //Annu. Rev. Phytopathol. – 2000. – Vol. 38. – P. 461-490.

3. JENNS A.E., DAUB M.E., UPCHURCH R.G. Regulation of cercosporin accumulation in culture by medium and temperature manipulation //Phytopathology. – 1989. – Vol.79, 1 2. – P. 213-219.

4. RUPPEL E.G. Variation amond isolates of Cercospora beticola from sugar beet //Phytopathology. – Vol. 62, No. 1. – 1972. – P. 134-136.

5. WHITNEY E.D., LEWELLEN R.T. Physiological races of Cercospora beticola on Beta vulgaris //Annual Proceedings of American Phytopathological Society for 1974. – 1974. – Vol. 1. – P. 88.

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